Chem.-Biol. Interactions, 35 (1981) 363--367 363 © Elsevier/North-Holland Scientific Publishers Ltd.

Short Communication

NICOTINE TOXICITY IN CHICK VERTEBRAL CHONDROCYTES IN VITRO

M.A. KHAN a, D.V. PROVENZA a, N.O. OLSON b and D.O. OVERMAN c

aBaltimore College of Dental Surgery, Dental School, University of Maryland at Baltimore, Baltimore, MD 21201, b Division of Animal and Veterinary Science and CDepartment of Anatomy, West Virginia University, Morgantown, WV 26506 (U.S.A.)

(Received February 9th, 1979) (Revision received December 18th, 1980) (Accepted December 22nd, 1980)

I n t r o d u c t i o n The cartilaginous vertebrae in the developing chick embryo appear

particularly sensitive to nicotine; the vertebral columns of the treated embryos are abnormally distorted with fusion of the articular surfaces and a decrease in size of individual vertebrae [1,2]. It is interesting to note that the chondroblasts in the vertebral bodies of the treated chick embryos, though disorganized and lacking in metachromatic matrix, appear normal morpho- logically [2]. It is, therefore, not clear if the primary effect of nicotine in chick embryos is on the differentiating chondroblasts leading to defective chondrogenesis or indirectly on associated myoblasts as suggested by Landauer [1]. Though, various investigators have reported on nicotine induced cytopathology in cultured fibroblast, mesenchymal and endothelial cells [3--6], the effect of nicotine on cultured chondroblast has not been investigated. In view of the above the present investigation was undertaken to determine morphological changes in vertebral chondroblasts treated with nicotine sulfate in vitro.

Materials and M e t h o d s The vertebral columns of 10-day embryos were diced into 1--2-mm pieces

and a cell suspension obtained by trypsinization (0.25% Trypsin, GIBCO). The dissociated cells were seeded in 75~m 2 plastic tissue culture flasks (Falcon) at a density of 20 × 106 cells/flask and incubated at 37°C in a humid atmosphere of 5% CO2. The culture medium consisted of MEM (GIBCO) supplemented with 10% fetal calf serum (GIBCO) and 1% anti- biotic-antimycotic solution (GIBCO). After 8 days the cells were trypsinized and secondary cultures prepared in Leighton tubes at a density of 1 X 106 cells/tube. Forthy-eight~hour secondary cultures were exposed to nicotine

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by replacing the culture medium with one containing 312, 156, 78, or 38 ~g/ml nicotine sulfate (40%, Sigma). The medium of some treated cultures was supplemented with 100 ~g/mg sodium ascorbate (GIBCO). The controls consisted of untreated cultures. After incubation for 24 h and 48 h, cells attached to the glass coverslips were briefly washed in PBS and stained with 1,9<limethylmethylene-blue for light microscopic observation. ,For ultrastructural, studies, treated cells were fixed in situ in 4% gluteraldehyde for 24 h at 4°C. The cells were then washed in Millonig's buffer (pH 7.3), postfixed in 2% osmium tetroxide, dehydrated in a graded series of alcohols and embedded in a epon-araldite mixture. This sections of chondrogenic areas were stained with alcoholic uranyl acetate and lead citrate.

Results Nicotine sulfate dose of 312 pg/ml was extremely toxic to the cultured

cells. Within 6 h of treatment the cells started to 'round up' and after 24 h a majority of the cells had detached from the cover glass. Nicotine doses of 156 pg/ml and 77 #g]ml did not effect cell death after 24 h of treatment through most cells appeared to be affected as indicated by vacuolar degene- ration. Forty~ight hours after treatment with 156 ug/ml of nicotine between 45% and 50% of the cells appeared moribound, while the remaining cells demonstrated large intracytoplasmic vacuoles. Nicotine dose of 77 ~g/ml induced cell pathology in the treated cultures in a manner similar to that observed in the 156-~g group, however, the cell mortality was less severe. The cells treated with 38 ~g/ml were only mildly affected and demonstrated numerous chondrogenic areas with metachromatic inter- cellular material. The polygonal cells in the middle of the chondrogenic colonies revealed a normal light and ultrastructural morphology while numerous small vacuoles were present in the cells at the periphery of the colony, many of which contained cytoplasmic material. Presence of sodium ascorbate (100 pg/ml) in the medium reduced the toxicity of nicotine sulfate at all doses of the drug. In the 312-~zg/ml and 156-~g/ml groups, though cell death was reduced by supplementing the medium with ascorbate, the degenerative vacuoles were still abundantly present in the cytoplasm. The chondrocyte cultures exposed to the lower doses of nicotine (77 ~g/ml and 38 ~g]ml) and cotreated with 100 pg/ml sodium ascorbate remained healthy throughout the culture period.

Ultrastructure of the untreated cultures demonstrated chondroblasts embedded in abundant extracellular matrix and containing prominent RER, juxtanuclear Golgi and occasionally a few small vacuoles (Fig. 1). In the cultures treated with 156 pg/ml nicotine there was a paucity of extracellular material and cell organelle. The chondroblasts appeared to be in various stages of degeneration demonstrating extensive vacuolization and systematic fragmentation of cell organelle (Fig. 2). Some vacuoles appeared empty while others contained an electron dense material which under closer examination was identified as remnants of degenerating cytoplasmic

Fig. 1. Chondrobhsts cultured for 72 h in control MEM. The cells demonstrate inter- cellular matrix and numerous cytoplasmic organelles. Orig. mag. ×19 000.

Fig. 2. Vacuolar degeneration in a 72 h chondroblast culture treated with 156 ~g/ml nicotine sulfate. Orig. mag. ×12 000. Fig. 3. Chondroblast culture (72-h) treated with 38 /zg/ml nicotine sulfate. A few degenerating vacuoles are present in some cells (arrow) while others appear normal. Orig. mag. ×6000.

Fig. 4. A higher magnification of a 72-h culture treated similar to the one in Fig. 3. The cells demonstrate normal extracallular matrix (t~M), however, the mit~chondria (M) appear swelled and the Golgi complex (G) is reduced to a few localized areas. Orig. mag. )<12 500.

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organelle. The cell membrane of a majority of cells appeared intact, however, in dying cells or cells affected with severe degeneration some vacuoles had ruptured through the cell membrane releasing the cytoplasmic organelle in the extracellular space. I n the cultures treated with 38 ~g of nicotine sulfate, though, the cells appeared normal (Fig. 3), subtle changes were observed in the cytoplasmic organelle. The mitochondria showed abnormal swelling; the Golgi complex though maintaining normal morphology was reduced to a few localized areas and there was a distinct paucity of RER (Fig. 4).

Discussion Nicotine is one of the most toxic drugs; it acts at a rate comparable to

that of cyanide poisoning [7,8]. Even though the drug has no therapeutic value, its high toxicity and presence in tobacco give it a considerable measure of importance in medicine. Thus there are many studies on its pharmacologic effects [5,7--9] however, only a few studies have been reported on its effects on developmental events [1,2]. Chondroblasts in the cartilaginous vertebrae of chick embryos injected with nicotine sulfate maintain normal morphology, though, the vertebral column demonstrates gross abnormal curvatures of scoliosis and spondylolisthesis. Ultrastructure of in vivo nicotine-treated chondroblasts has not been investigated. The present study, demonstrates that under in vitro conditions the sensitivity of cultured chondroblasts is dosage dependent. At 312-gg/ml dose, cell death was the major consequence of nicotine; at lower doses {156, 77 and 38 pg/ml) the drug expressed its cytotoxicity by inducing a progressively increased cellular breakdown and thus an overall reduction of all synthetic activities concomittant with an increase in culture age and drug dose. The basic mechanism of nicotine sulfate-related teratogenic effects may be similar to the degenerative processes described by Herrmann [10], who observed cellular breakdown and resorption after the administration of leucine antagonists. It was interesting to note that the cells treated with 38 pg/ml nicotine sulfate demonstrated normal ultrastructural morphology, however, at higher magnification changes in the cell organelle were evident. Not all cells were affected similarly; this was especially evident in cultures treated with higher doses of nicotine where degeneration in the chondro- genic areas was localized in necrotic foci with adjacent cells which appeared relatively normal. Previous investigators have questioned the ascorbate requirement for growth of cultured cells [11,12]. The present investigation and those reported by others [13,14] indicate the beneficial effects of ascorbate in maintaining the structural integrity of chondroblasts and the intercellular matrix. In the present investigation, though cytoplasmic vacuoles were still present, the presence of ascorbate greatly reduced pyknosis and karyorrhexis in cultures treated with higher doses of nicotine. Cultures treated with 77-~g/ml and 38-~g/ml doses of the drug were completely protected with addition of sodium ascorbate in the medium. The mechanism by which the vitamin exerts its beneficial effects in vitro is

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not clear. The present investigation supports the suggestion by Landauer [ 1 ] that sodium ascorbate, because of its role as an intermediate metaboli te is able to provide the cells with an alternate, exogenous, source of energy. However, confirmatory evidence is needed and in vitro studies utilizing monolayer cultures should provide an ideal system for such investigations.

1 W.J. Landauer, Nicotine-induced malformations of chicken embryos and their bearing on the phenocopy problems, J. Exp. Zool., 143 (1960) 107.

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8 K.E. Jackson, Alkaloids of tobacco, Chem. Rev., 29 (1941) 123. 9 S.J. Konturek, T.E. Solomon, W.G. McCreight, L.R. Johnson and E.D. Jacobson,

Effect of nicotine on gastrointestinal secretions, Gastroenterology, 60 (1971) 1098. 10 H.J. Herrmann, Interference of amino-acid analogues with normal embryonic develop-

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14 J.R. Schlitz, J. Rosenbloom and G.E. Levenson, The effects of ascorbic acid deficiency on collagen synthesis by mouse molar tooth germs in organ culture, J. Embryol. Exp. Morphol., 37 (1977) 49.